Ion mobility spectrometry (IMS) comprises a family of techniques for separating ions on the basis of their mobility in the gas phase. Ion drift mobility (IDM) spectrometry was introduced as an analytical technique in the beginning of the 1970's, although ion mobility research had been carried out for several decades previously, mostly for ion characterisation. In the 1980's, the first analytical instrument based on field-asymmetric ion mobility spectrometry (FAIMS) principles was described.
Throughout this document, IMS will refer to all techniques that have the capability of separating ions in the gas phase on the basis of their ion mobility characteristics. One such technique is “classical” ion drift mobility (IDM) spectrometry in which ion separation occurs in a static, homogeneous axial electric field. By contrast, another such technique, FAIMS, is based on an asymmetrical time-alternating electric field, which is transverse to the net direction of ion motion in a flowing bath gas. These features will be clarified before presenting the scope of the instant invention.
In ion drift mobility spectrometry, pulses of ions (ion “packets”) are typically introduced into a gas-containing tube with a static, axial, and ideally constant electric field E that drags the ions through the bath gas. An ion packet composed of ions characterized by mobility K will “drift” with constant speed v=K·E (with a small correction for diffusion). If the ion packet contains components characterized by differing ion mobility values K, these components acquire different speeds. Consequently, the ions are separated according to their ion mobility values. When recording the time profile of the intensity of ions arriving at a detector after introducing an ion packet into the drift tube at a well-defined time, an ion mobility spectrum is obtained. This ion mobility spectrum reflects the range of mobility values possessed by the ions comprising the ion packet. Mobility also depends on an ion's charge state. Of greater significance and utility, mobility depends on the detailed interactions between analyte ion and bath gas, as reflected by the orientationally-averaged collision cross section. Thus, the range of mobility values observed in an IDM experiment can reflect different conformations of the same ion (e.g. same chemical formula and covalent connectivity, as in proteins of different conformation); different structures of constitutional isomers (e.g. same chemical formula but different covalent connectivity as in carbon clusters); or different arrangement in space of the atoms of diastereomers; or it can reflect the presence of a number of completely different ions (e.g. ions of different structures and compositions). Normally, but not necessarily, electric fields in IDM are relatively weak, so that ions have an ion mobility K=Ko that is independent of the electric field strength E.
In its most widespread design, an IDM instrument features a drift tube with evenly-spaced concentric drift rings carrying different voltages along the interior of the drift tube, ensuring the presence of a constant axial electric field of negligible inhomogeneity along and near the central axis.
FAIMS exploits the fact that, at sufficiently high electric fields, most ion species display a dependence of their ion mobility on the electric field strength—the mobility is no longer electric-field-independent, K=K(E). If we express K(E) as K0·(1+h[E]), the function h(E) will vary depending on the structure and identity of the ion. FAIMS separates ions characterized by differing ratio of high-field mobility to low-field mobility—i.e. ions are separated according to the quantity 1+h(E). This is done by alternating a high electric field of one polarity during a short time, with a low electric field of the opposite polarity during a longer time. In such a scheme, ions would oscillate and return to their starting positions after each cycle, if it were not for the dependence of the ion mobility on the electric field strength. This dependence gives each ion a net non-zero displacement during each cycle. Typically, FAIMS separation takes place between two parallel plates or between two concentric cylinders with a spacing of a few millimeters. An axial (or longitudinal) gas flow sweeps the ions along a path roughly parallel to the axial direction, while the applied electric field causes transverse ion motion roughly perpendicular to the gas flow. When cylindrical electrodes are employed, FAIMS features an additional ion focusing effect because the electric field between the cylinders possesses a gradient.
FAIMS separation is driven by the dispersion voltage (DV), a high-voltage asymmetric waveform that is applied between the two electrodes. By applying an additional constant but adjustable compensation voltage (CV), the net displacement of a certain ion species during each DV waveform cycle can be compensated, resulting in those ions being transmitted through the FAIMS while non-compensated ions are lost in collisions with the electrode surfaces. By scanning CV and measuring the transmitted ions at the ion outlet, data can be obtained in the form of a “CV spectrum”. Alternatively, the CV can be kept constant or can be cycled over a few predetermined values, so as to only transmit ions of interest.
Both IDM spectrometry and FAIMS can be operated at different gas pressures, gas temperatures and gas compositions. Both IDM spectrometry and FAIMS have been proposed and/or shown to work with different ion sources, including electrospray ionisation (ESI), nanospray ionisation (nESI), ionspray, atmospheric pressure chemical ionisation (APCI), matrix-assisted laser desorption-ionisation (MALDI), and beta emitter ionisation. Other ionisation techniques could be used as well. Both IDM spectrometry and FAIMS have been coupled to different detectors, such as mass spectrometers (MS), and charge-measuring devices like a Faraday plate coupled to an electrometer. One big difference between IDM spectrometry and FAIMS is that IDM spectrometry typically is a pulsed technique, relying on an ion flight time to link a mobility value with a particular sub-population of ions, while FAIMS is a continuous-flow technique that can be set to allow continuous passing of a distinct population of ions characterized by a certain relation between high-field and low-field mobility. Ion drift mobility spectrometry and FAIMS principles and applications are described in exhaustive detail by Eiceman and Karpas in their book “Ion Mobility Spectrometry”, 2nd Edition (2005), CRC Press, Boca Raton, Fla.
Different structural isomers and stereoisomers of molecules often feature different physical, chemical and biological properties. It is therefore of importance that analytical chemistry promote the development of tools to distinguish between different isomers. One group of stereoisomers, the enantiomers, is of specific interest because two enantiomers have different chemical properties only in asymmetric environments. Life itself offers one of the key examples of asymmetric environments—and many cases are known for which two enantiomers have very different effects on biological systems. One example is drug substances where one enantiomer has a therapeutic effect while the other enantiomer has an adverse effect. It is a challenge for analytical chemists to be able to develop techniques that can either separate enantiomers or else reliably detect one such enantiomer in the presence of another—even in cases where there is a large concentration difference.
Another area in which isomers are of key importance is carbohydrate chemistry. Carbohydrate monomers exist as a large variety of structural isomers and stereoisomers, and carbohydrate oligomers and polymers display an even greater variety. To understand the relationship between structure and property of carbohydrates, and to be able to develop and manufacture new products from carbohydrates like starch and cellulose, new analytical techniques are needed that can distinguish between different isomers. For example, the separation and analysis of sugar dimers of the same mass, but of different stereochemistry, poses a great challenge when employing existing techniques.
The most widely employed approach for separation of enantiomers is liquid chromatography (LC) with chiral stationary phases. Capillary electrophoresis, capillary electrochromatography and gas chromatography are also employed in chiral separations. While useful in many cases, chromatography is often time-consuming. The chiral phases employed are also said to be “less rugged”. Another approach for determining enantiomeric excess in samples is NMR. Some kind of chiral shift reagent is required, along with fairly copious amounts of sample. Although useful for some problems, none of these techniques comprises a general solution for the analysis of enantiomers.
Guevremont et al. (WO 00/08454) (“Guevremont”) propose a “method for separation of isomers and different conformations of ions in gaseous phase” using FAIMS. Analytes are ionised and subjected to FAIMS separation. Guevremont includes enantiomers in their list of analytes that can be separated with the aid of FAIMS; however, they do not give any examples for separated enantiomers. It seems doubtful that their method alone can separate enantiomers, as enantiomers can only be separated in an asymmetric chemical environment, and their method lacks such asymmetric elements.
Tao et al. (Analytical Chemistry, Vol. 71, No. 19, 1999) (“Tao”), and others, have proposed a mass spectrometric method that leads to the quantification of enantiomers by providing just such an asymmetric environment. In this method, trimeric cluster ions containing one metal ion, two molecules of an enantiopure reference compound, and one molecule of a chiral analyte compound are formed by association in solution phase. Cluster ions containing one enantiomer of the analyte versus the other enantiomer of the analyte are considered to be diastereomeric. These cluster ions are electrosprayed and subjected to tandem (fragmentation) mass spectrometry. From the relative intensities of certain of the formed fragment ions, conclusions can be drawn about the enantiomeric excess of one enantiomer analyte over the other, when the reference compound and metal ion have been suitably selected. Often a notable chiral sensitivity can be achieved. This method does not lead to a physical separation of diastereomeric cluster ions prior to mass analysis, since both diastereomeric cluster ions are detected at the same time and mass-to-charge ratio. Rather, the relative amount of the enantiomer analytes is reflected indirectly in the fragmentation pattern through the energetics of fragmentation of the diastereomers. Advantages of the technique include its simplicity, the need for only small amounts of material, and the tolerance of the method to the presence of background impurities. One drawback is the relatively high uncertainty of the measurement, typically at one or a few percent of enantiomeric excess, limiting the usefulness of the method in case very small amounts of one enantiomer are present in a large excess of the other enantiomer.
Different approaches combining host-guest systems with mass spectrometric measurements in order to analyse enantiomers have been described as well. Generally, in this method, a large chiral molecule (host) interacts non-covalently with a small chiral analyte (guest) in a stereoselective way. Mass spectrometric determination of the enantiomeric excess is then performed e.g. with the help of isotope labelled/unlabelled chiral host pairs and isotope labelled internal standards (e.g. Shizuma et al., Int. J. Mass Spectrom. 2001, 210/211, 585-590), or by stereoselective gas-phase exchange reactions (e.g. Grigorean et al., Anal. Chem. 2000, 72, 4275-4281).
Karas (WO 02096805) (“Karas”) proposes an ion drift mobility spectrometric method for separating enantiomers and other isomers and compounds, “using a supply of selectively interactive gaseous particles” as “separating substance”. For the case of enantiomer separation, in the first preferred embodiment of the patented idea, chiral gaseous molecules are added to the bath/drift gas for collisions, which undergo clustering/declustering interactions with the chiral analyte ions. In case one enantiomer has a stronger interaction with the neutral chiral collision molecules (i.e. separation particles) than the other enantiomer, the retention time of the first enantiomer in an IDM experiment would be prolonged, because said first enantiomer would presumably display a higher time-averaged collision cross section due to clustering with the chiral gas molecule. In the second preferred embodiment of the patented idea, the gaseous particles used for selective interactions with the enantiomers (or other compounds) are macroscopic particles such as macromolecules or nanoparticles, possibly ones with smaller chiral molecules bound to said particles surfaces, intended to provide a local chiral environment to which enantiomers may be bound in interaction processes for different periods of time depending on their chirality. These particles are added into the bath/drift gas as described just above. Again, the separation according to this invention relies on differences of the affinity or timescale of interaction between the two enantiomers and the separating particles as sampled via collisional interactions, driving a difference in drift time that separates the chiral enantiomers. It is a characteristic of the patented idea that such “separating substances”, “which comprises gaseous particles which selectively interact with the molecules of the components to be separated”, are in fact not present in the sample, but are supplied separately to the bath gas, in order to selectively interact in collisions with the analytes of interest. It is characteristic for Karas' invention that the reference substance is provided to the IDM apparatus continuously, as long as analytes are present in the apparatus. In Karas' invention, all interactions between analytes of interest and the separating substance have the nature of collisions or temporary short-lived associations. Unfortunately, it is not obvious that a useful degree of separation has taken place in the example presented in Karas' patent.
Leavell et al. (J Am Soc Mass Spectrom 2002, 13, 284-293), and others, show the separation of diastereomers by IDM, without the addition of chiral reference compounds.
A number of techniques are used in order to separate or analyse constitutional isomers and different forms of stereoisomers, e.g. liquid chromatography and NMR. However, new approaches capable of distinguishing between isomers are still of importance.
It is a limitation of the prior art that small amounts of one enantiomer in presence of a large excess of the other enantiomer cannot be rapidly determined using a small amount of sample.
It is a limitation of the prior art that constitutional isomers and stereoisomers often are not readily separable with existing techniques.